DESCRIPTION: The long-range goal of this proposal is to establish Saturation-Recovery Pulse EPR spectroscopy as a significant experimental modality in the determination of biomolecular structure and dynamics. Specific Aims designed to meet these goals are : 1, development of bimodal loop-gap resonators; 2, design and construction of a new x-band saturation recovery spectrometer that will provide a platform from which the PI can continue instrumentation development while utilizing existing spectrometers for applications; 3, application of SR-ESR methods to accurately measure distances between spin labels in site directed spin labeling (SDSL) studies ; and 4, application of SR-ESR and pulse ELDOR studies to understand the non-exponential response of Cu(II) proteins and Cu(II) model complexes. Attention will be given to improving the reliability of measurements of spin-lattice relaxation times. These measurements are key to distance determinations. Site-directed spin labeling using the methodology of molecular biology is a powerful approach to the determination of structure of membrane-bound peptides and proteins. Saturation recovery and pulse ELDOR are experimental methods of choice for determination of bimolecular collisions of oxygen and paramagnetic metal ions with spin labels, which is a major aspect of SDSL methodology. Studies on copper are motivated by anomalous spin-lattice relaxation results from cytochrome-c oxidase and nitrous oxide reductase and are directed at understanding the anomaly and improving the quality of the data. Instrumental development proposed here can contribute to instrumental design in other fields of spectroscopy including spin-echo EPR and high-resolution NMR.